Infection control is critical to the safety of laboratory scientists who work with infectious diseases. With this in mind, we don our personal protective equipment (PPE), which is expensive to maintain and has diminishing utility when contaminated with pathogens, and continuously disinfect our workspace—using alcohols, chlorine compounds, such as bleach, ammonium compounds, phenolics and even hydrogen peroxide in some settings. Each of these has its own properties, which facilitate elimination of particular microbes on the bench, but they are also all notoriously hazardous if used improperly or without training.

The need for additional disinfectants is clear, and in Dec. 2020, a team of researchers published their findings on the use of methylene blue (MB) and light to decontaminate SARS-CoV-2 on personal protective equipment. MB decontamination has been periodically explored in dentistry settings since the 1990s. However, this most recent (and somewhat surprising) discovery began a cascade of new research exploring other potential use cases for MB decontamination against a variety of pathogens. This growing area of research has profound implications for the paradigm of infection control in both clinical and laboratory settings.

How Does Methylene Blue Disinfect?

Methylene blue, also known as “methylthioninium chloride,” is a positively charged dye that is inherently attracted to acidic cell components, such as the cell nucleus, and a photosensitive compound that releases singlet oxygen (¹O₂) when exposed to visible (ambient) light. Singlet oxygen, a kinetically unstable molecule that is also produced by the human body, is highly reactive toward organic compounds, including virus membranes and genetic material. Many viruses, including coronaviruses, are encased in a lipid bilayer membrane. When this membrane comes in contact with reactive oxygen species like singlet oxygen, the resultant oxidation reaction alters the membrane's structure and damages its integrity, exposing cellular contents, namely genetic material, to further oxidation reactions which can damage nucleotide bases and/or cause strand breakage.

A growing body of research shows that photochemical treatment of surfaces with MB is highly effective at killing pathogens such as SARS-CoV-2, Ebola virus, Middle East respiratory syndrome coronavirus (MERS), Crimean-congo hemorrhagic fever virus (CCHFV),  Nipah virus, Streptococcus mutans and, most recently, norovirus. Other applications outside of PPE decontamination have yet to be explored.

Traditional Use of Methylene Blue

MB is useful in a variety of hospital and laboratory settings, and across disciplines such as hematology, immunohematology and clinical microbiology.

In hospital settings, MB is commonly used to treat the blood disorder methemoglobinemia. Methemoglobinemia is characterized by an abnormally high volume of methemoglobin in the blood; this can be caused by certain medicines or foods, or it can be congenital. Unlike hemoglobin, methemoglobin cannot transport oxygen; therefore, increases in methemoglobin can lead to hypoxia. At lower levels, symptoms include dyspnea, nausea and tachycardia. At higher levels, symptoms can evolve to lethargy, stupor, deteriorating consciousness, cardiac arrhythmias and death. When low doses of MB are injected as treatment, the body’s metabolism reduces MB to leukomethylene blue, which is a reducing agent that reacts with methemoglobin and lowers overall methemoglobin levels in the bloodstream.

MB also has a treasure trove of uses in the laboratory. In hematology, MB is used in Wright’s and Jenner’s stains to differentiate blood cell types. Staining facilitates visualization of white blood cells and differentiation based on color and morphology. In microbiology, MB is useful to observe pathogen genetic material under a microscope. When MB comes in contact with a pathogen’s nucleic acid (which, as the name suggests, is slightly acidic), the resulting redox reaction causes the RNA or DNA to turn a characteristic blue color, making it easier to observe. MB staining is also commonly used in northern blotting procedures to detect and measure expression of predefined strands of RNA.

MB has been sporadically explored as a decontaminant since the early 1990s. However, this research focused on periodontal disease in dentistry. Specifically, one study impregnated teeth with MB (post-extraction), exposed them to light and found a reduction in the number of Streptococcus mutans colonies compared to control groups. Research then shifted into transfusion medicine applications in the late 1990s, and MB is now used to decontaminate plasma products prior to transfusion in multiple European countries by dissolving an MB tablet in thawed fresh frozen plasma, exposing it to light, then transfusing. Exploration of MB as a healthcare (and PPE) decontaminant only came about in 2020.

The Serendipitous Discovery

Before diving into the disinfection qualities of MB, it is important to discuss the intent and history of the project that led to the discovery. Early during the SARS-CoV-2 pandemic, supply shortages forced health care workers to re-use their PPE. To try to improve the quality of PPE disinfection for reuse, the World Health Organization (WHO) led the "Development of Methods for Mask and N95 Decontamination" (DeMaND) Study. DeMaND was carried out by 52 scientists from 13 different universities and labs. These researchers originally sought to explore dry heat as a PPE decontamination method, but they also introduced MB with light as an exploratory method along the way. The hallmark research (published in 2021) demonstrated that MB treatment of respirators and other PPE commonly used in a healthcare setting inactivated 98.9 to >99.9% coronaviruses following 5 to 30 minutes of light exposure, depending on the concentration of MB.

PPE decontamination protocols, particularly those that are safe, inexpensive and non-invasive, are greatly needed for frontline healthcare workers. This discovery primarily impacts healthcare workers in lower resource settings, where PPE reuse for resource management has historically led to increases in healthcare worker infections, and in the case of Ebola, healthcare worker transmission of disease. The use of MB decontamination for PPE (for up to 5 uses) could be particularly impactful in these areas, and for frontline healthcare workers in general. Importantly, MB decontamination does not compromise the integrity of PPE like other decontamination procedures, such as heat treatment. Healthcare workers can therefore maintain the integrity of their PPE while keeping themselves safe from infection in the process.

 

The success of DeMaND study prompted WHO to add MB decontamination procedures to their overall guidance on “Rational use of personal protective equipment for coronavirus disease (COVID-19) and considerations during severe shortages.” The success also prompted a DeMaND-2 follow-up study, which is currently exploring applications of MB decontamination for other high-threat pathogens. MB disinfectant is an extremely stable, harmless and cost-effective alternative to other common disinfectants used in the laboratory, such as bleach or alcohol. Going further, the formulation process is very user friendly. You only need MB salt or 1% aqueous MB solution, deionized water and an aluminum-cased spray bottle. As future MB decontamination research takes flight, it will be interesting to see how additional regulatory bodies integrate this application into new standards, and how this new discovery will positively impact infection control and public health around the world.